PPP6C, a serine-threonine phosphatase, regulates melanocyte differentiation and contributes to melanoma tumorigenesis through modulation of MITF activity

It is critical to understand the molecular mechanisms governing the regulation of MITF, a lineage specific transcription factor in melanocytes and an oncogene in melanoma. We identified PPP6C, a serine/threonine phosphatase, as a key regulator of MITF in melanoma. PPP6C is the only recurrently mutated serine/threonine phosphatase across all human cancers identified in sequencing studies and the recurrent R264C mutation occurs exclusively in melanoma. Using a zebrafish developmental model system, we demonstrate that PPP6C expression disrupts melanocyte differentiation. Melanocyte disruption was rescued by engineering phosphomimetic mutations at serine residues on MITF. We developed an in vivo MITF promoter assay in zebrafish and studied the effects of PPP6C(R264C) on regulating MITF promoter activity. Expression of PPP6C(R264C) cooperated with oncogenic NRAS(Q61K) to accelerate melanoma initiation in zebrafish, consistent with a gain of function alteration. Using a human melanoma cell line, we examined the requirement for PPP6C in proliferation and MITF expression. We show that genetic inactivation of PPP6C increases MITF and target gene expression, decreases sensitivity to BRAF inhibition, and increases phosphorylated MITF in a BRAF(V600E) mutant melanoma cell line. Our data suggests that PPP6C may be a relevant drug target in melanoma and proposes a mechanism for its action.

www.nature.com/scientificreports/ Regulation of the activity and expression level of MITF is in part controlled by its phosphorylation 13 . KIT-mediated ERK signaling can phosphorylate MITF, leading to an increase in its expression 14 . The phosphatases that target MITF for dephosphorylation remain unknown. In melanoma, MITF can function as a lineage survival oncogene, where unregulated expression can transform melanocytes 15 . However; MITF expression varies across melanomas, producing different melanoma phenotypes 16 . At lower levels of expression, MITF upregulates other transcriptional programs such as increasing expression of DIAPH1 leading to suppression of CDKN1B 17 . In melanomas, an MITF-low protein level is associated with invasion and proliferation 18 and has been shown to be relatively resistant to immunotherapies 19 . Therefore, modulation of MITF during melanoma tumorigenesis and progression remains an important area of study.
In this study, we have defined a novel function for PPP6C during melanocyte development and implicated PPP6C(R264C) in accelerated melanoma tumorigenesis. Using genetic approaches, we have explored the consequences of gain and loss of PPP6C function on melanocyte specification, proliferation, and the development of melanoma in vivo. These data suggest that the phosphatase PPP6C blocks melanocyte differentiation and the recurrent R264C mutation is a gain of function alteration that leads to enhanced tumorigenesis.

Results
PPP6C is recurrently mutated in melanoma and has a unique relationship with the melanocyte lineage and MITF. The Cancer Genome Atlas Project performed next generation sequencing on 331 primary and metastatic melanomas. Mutations in PPP6C were identified in 7% (n = 24) of tumors sequenced. Of those, 36% (n = 9) harbored a R264C mutation. PPP6C(R264C) co-occurred with both BRAF(V600E) and NRAS(Q61K) mutations 5 . We further analyzed PPP6C mutation data from 48,000 tumors across 184 pan-cancer studies. This analysis reveals that PPP6C mutations were identified in 285 tumors. Overall, 22% (n = 58) of PPP6C-mutated tumors contained the hot-spot PPP6C(R264C) mutation. All but one of these tumors containing a PPP6C(R264C) mutation were melanomas, suggesting a unique requirement for PPP6C in melanocytes (Fig. 1A). PPP6C appears to be the only recurrently mutated serine threonine phosphatase across all human cancers 20 , suggesting it plays an important role in melanoma regulation.
We leveraged publicly available CRISPR loss of function screens across a diverse array of human primary and immortalized cell lines to investigate the requirements for PPP6C in proliferation 21 . Melanoma cell lines are uniquely dependent on PPP6C expression for proliferation (Fig. 1B). Based on this data, we suspected a unique relationship between PPP6C and the melanocyte lineage. MITF is the critical transcriptional regulator of melanocyte development and upregulates a set of genes to drive differentiation 11,12 . In order to explore whether there was a link between the transcriptional program of MITF and PPP6C, we investigated 42 publicly available melanoma cell lines from the Broad Institute 22 . Our analysis reveals that PPP6C expression negatively correlates with MITF target gene expression (Fig. 1C). These findings suggest that PPP6C may play a previously unexamined role in MITF regulation in the melanocyte lineage.

PPP6C disrupts melanocyte differentiation in vivo.
In order to investigate whether PPP6C regulates MITF in vivo we utilized genetic approaches in zebrafish. Using CRISPR, we performed targeted disruption of the PPP6C coding sequence in wild-type embryos. Targeted disruption of ppp6c led to embryonic lethality at 24 h post fertilization (Supplemental Fig. 1). This is consistent with prior research in mice showing the requirement of PPP6C for post implantation embryogenesis 8 . In order to study PPP6C specifically in the melanocyte compartment, expression via the MiniCoopR vector was used. The tol2 transposon-based MiniCoopR vector was used to express the human orthologue of PPP6C as well as the zebrafish orthologue of MITF (mitfa) open reading frame under control of the mitfa promoter (as described in Iyengar et al. 23 ). The resulting vector was then injected into 1 cell stage embryos containing a mutation in mitfa 24 . Therefore, only fish in which the vector was successfully incorporated underwent melanocyte differentiation, allowing us to examine the effects of PPP6C expression in melanocytes. Vectors encoding PPP6C, PPP6C(R264C) and GFP (control) were created (referred to as MC-PPP6C etc.). Expression of PPP6C reduced the number of melanocytes on 5 day old fish as compared to GFP controls ( Fig. 2A,B). Expression of PPP6C(R264C) further reduced the number of melanocytes. These fish were assayed for mitfa and target gene expression. While neural crest markers were unaffected, mitfa and mitfa target gene expression was reduced in fish expressing PPP6C and further reduced in fish expressing PPP6C(R264C) (Fig. 2C,D). These results indicate that in vivo expression of PPP6C affects melanocyte differentiation and results in a phenotype of fewer melanocytes.
Prior research has shown that regulation of the phosphorylation status of MITF is important for its expression and activity, as different transcriptional programs are upregulated based on MITF levels. Phosphorylated MITF, regulated in part by KIT-mediated phosphorylation, is active, increases its own expression and upregulates a transcriptional program of melanocyte differentiation 16,25 . With this in mind, we hypothesized that there was an interaction between PPP6C and Mitfa, and used genetic approaches in zebrafish to test this hypothesis. Prior loss of function research has shown the serine residues S69 and S73 on MITF play a role in melanocyte differentiation 14,25,26 . These two residues are conserved in the zebrafish orthologue as S8 and S12 (Fig. 3A). We performed site-directed mutagenesis on the mitfa open reading frame to selectively mutate S8 and S12 to phosphomimetic aspartic acid residues (hereafter referred to as "mitfa(S8D,S12D)"). Injection of vectors containing mitfa(S8D,S12D) mutations resulted in a rescue of melanocyte number in fish expressing PPP6C or PPP6C(R264C) (Fig. 3B,C). At the molecular level, introduction of a phosphomimetic S8D,S12D mitfa also rescues mitfa and mitfa target gene expression (Fig. 3D,E). Together, these experiments indicate that PPP6C impacts Mitf phosphorylation during differentiation and the R264C mutation has an impact on melanocyte differentiation 27  www.nature.com/scientificreports/ MITF promoter activity is reduced under expression of PPP6C(WT) and PPP6C(R264C). In order to directly study the effect of PPP6C on mitfa gene expression we developed a reporter gene assay in transgenic zebrafish embryos. MITF is able to transactivate its own promoter and participates in a positive feedback loop to maintain expression 28,29 . To measure mitfa promoter activity, we utilized the MiniCoopR encoding GFP. MiniCoopR vectors containing GFP and either PPP6C(WT) or PPP6C(R264C) were injected into fish containing albino and mitfa mutations, resulting in fluorescent and pigment less melanocytes, (Fig. 4). Melanocytes were imaged and fluorescent intensity was quantified to measure mitfa promoter activity. The effects of PPP6C on mitfa promoter activity were measured first on a non-oncogenic background. As compared to GFP controls, embryos containing either PPP6C(WT) or PPP6C(R264C) showed reduced GFP signal, consistent with reduced mitfa promoter activity (Fig. 4A). Next, we sought to determine if this reduced activity was consistent on both the BRAF and NRAS oncogenic backgrounds. Regardless of oncogenic background, melanocytes expressing PPP6C(WT) had reduced GFP expression and mitfa promoter activity (Fig. 4B,C). Additionally, embryos containing PPP6C(R264C) showed a further reduction in GFP expression and mitfa promoter activity. This data demonstrates that PPP6C has an effect on mitfa promoter activity in vivo, consistent with a negative effect on mitfa transcriptional activity. Additionally, the melanoma-specific oncogenic mutant PPP6C(R264C) led to a further reduction in expression of mitfa:GFP in vivo, suggesting that R264C is a gain of function mutation.
The recurrent R264C mutation confers a gain of function proliferation phenotype in melanoma. We sought to determine the impact the R264C mutation has on cellular proliferation and tumor onset in a melanoma model, since low MITF expression has been shown to be oncogenic in a subset of melanomas 17,18 and promotes tumor initiation in cultured melanoma cell lines 30 . Fish were injected with a vector co-expressing NRAS(Q61K) and either PPP6C(WT) or PPP6C(R264C). At 3 days and 5 days post fertilization, the num- www.nature.com/scientificreports/ ber of melanocytes on the head of the fish were counted (Fig. 5A). The percentage change in number of melanocytes between days was calculated. PPP6C(R264C) led to a significant increase in melanocyte proliferation as compared to expression of PPP6C(WT) (Fig. 5B). Zebrafish expressing PPP6C(R264C) and NRAS(Q61K) developed tumors significantly faster than those injected with wildtype PPP6C and NRAS(Q61K) (Fig. 5C). Together, this data points to the role of PPP6C as a modulator of mitfa expression and suggests that expression of PPP6C(R264C) enhances melanoma initiation on an NRAS(Q61K) background.

PPP6C expression affects MITF in melanoma.
After observing that mitfa transcriptional activity is negatively affected by PPP6C expression, we sought to identify the changes that might occur when PPP6C expression is reduced in an MITF-low melanoma. We hypothesized that genetic inactivation of PPP6C would lead to higher levels of MITF and promote a more differentiated cellular state. In order to test this hypothesis, we employed the A375 melanoma cell line, which expresses very low levels of MITF 31 . Cells were transfected with an siRNA against PPP6C to reduce gene expression (Fig. 6A). MITF and MITF target gene expression was assayed. PPP6C knockdown increased MITF and MITF target gene expression significantly (Fig. 6B). Additionally, we measured the effect PPP6C expression has on drug sensitivity. A375 cells were treated with dabrafenib, a small molecule inhibitor of BRAF(V600E) 32 and knockdown of PPP6C was performed (Fig. 6C). Cells treated only with dabrafenib or with a non-targeting siRNA displayed a robust response to treatment (EC50 = 0.3 nM). By comparison, cells treated with PPP6C siRNA showed a 15-fold decrease in sensitivity (EC50 = 4.6 nM). This data indicates PPP6C is able to modulate expression levels of MITF and induce drug resistance in a BRAF(V600E) mutant human melanoma cell line. Finally, we examined MITF expression and phosphorylation after PPP6C knockdown. We find no significant change in MITF protein expression, as determined by confocal microscopy after immunofluorescent staining after PPP6C knockdown (Fig. 6D). We do find an increase in phospho-MITF as measured by confocal microscopy after immunofluorescent staining of cells treated with PPP6C siRNA (Fig. 6E).

Discussion
Our data reveals a novel role for PPP6C in regulating the activity of MITF in melanocytes and melanoma. In  www.nature.com/scientificreports/ Phosphatases continue to emerge as important and druggable targets across multiple human cancers. SHP2, a tyrosine phosphatase involved in MAP kinase signaling acquires gain of function mutations such as D61Y that enhance its activity. These gain of function mutations are found in a variety of cancer types such as lung cancer, acute myeloid leukemia, and colon cancer 33 . Recently, SHP2 inhibitors such as SHP099 have shown promising efficacy in preclinical experiments and have entered clinical trials 34 . The recurrent R264C mutation in PPP6C may be another critical drug target as it is uniquely found in human melanoma.
Treatment of patients with BRAF(V600E) melanoma with BRAF and MEK inhibitors such as dabrafenib and trametinib have proven to prolong patient survival 2 . However, about half of patients do not respond to inhibition and show progression of disease. It has been shown that in some melanomas, reducing MITF expression can sensitize cells to chemotherapeutics 15 , consistent with the rheostat model of modulating MITF expression in  www.nature.com/scientificreports/ melanoma. Our results show that knockdown of PPP6C and the associated increase in MITF leads to decreased sensitivity to BRAF(V600E) inhibition (Fig. 6C). Regulation of MITF activity and phosphorylation are important melanoma initiation and progression. Higher levels of MITF expression are associated with cell cycle arrest and pigment-producing differentiation. As MITF levels decrease, programs associated with proliferation, survival, and invasion are upregulated; however, some baseline level of MITF expression is required to prevent senescence 35,36 . A growing body of evidence shows that in vivo, tumors switch between multiple MITF high and MITF low -associated phenotypes in response to changes in the environment including BRAF inhibition, hypoxia or inflammation [37][38][39] .
Based on the data presented we suggest that PPP6C plays a role in disease modulation and phenotypic heterogeneity through its regulation of MITF. Expression of the gain of function mutation R264C cooperated with BRAF and NRAS oncogenes to reduce mitfa expression and led to an increase in melanoma proliferation in a zebrafish model system. This is consistent with prior studies that have shown that MITF haploinsufficiency leads to cell cycle stimulation. Mice heterozygous for MITF produce fewer melanoblasts, but these cells display greater proliferation during migration 40 . Additionally, we demonstrate that genetic inactivation of PPP6C affects sensitivity to BRAF inhibition, suggesting that PPP6C levels may be predictive of clinical response to BRAF inhibitors. Finally, we find that genetic inactivation of PPP6C leads to a modest increase in phosphorylated MITF. Further  www.nature.com/scientificreports/ studies will be required to precisely identify the Ser/Thr residues targeted by PPP6C. Our data suggest that PPP6C plays a role in modulating MITF function as prior research has shown that MITF phosphorylation has an effect on both transcriptional activity and stability 26,41,42 . Together, these results define a unique role for PPP6C in melanocyte development and melanoma and place it upstream of regulating MITF expression and function.

Methods
TCGA data mining. All TCGA data sets for somatic mutations for cutaneous melanomas and other cancers were acquired from http:// www. cbiop ortal. org.
Preparation of transgenic vectors. All vectors were created using Gateway recombination (Life Technologies). A human PPP6C middle entry clone was made by PCR amplification as previously described 43 . pME-GFP, pME-PPP6C, pME- PPP6C  www.nature.com/scientificreports/ with 8 bits per pixel. A 405 and 640 laser were used to capture the DAPI channel and the 647 channel for each field respectively. The DAPI channel had a pinhole of 45 μm and was set to 8.0% laser intensity. The master gain was set to 700 V, while the digital offset was 0 and the digital gain was 1.0. The 647 channel had a pinhole of 41 μm and was set to 4% laser intensity. The master gain was set to 740 V, while the digital offset was 0 and the digital gain was 1.0.
Cell Titer Glo. Cells were plated at a concentration of 5000 cells onto 96 well plates and incubated at 1% FBS (Denville Scientific). Dabrafenib was purchased from Selleckchem. Cells were treated with dabrafenib at concentrations of 0.1-100 nM. At 48 h fresh drug was applied to cells. After 72 h cells were brought up to room temperature and 100 µL of CellTiter-Glo (Promega) reagent was added directly to each well. Plates were incubated on a shaker for 5 min and luminescence was measured on a Synergy 4 reader (Biotek). Luminescence readings were normalized to and shown as a relative percentage of DMSO control readings.
Zebrafish husbandry. Zebrafish were bred and raised in accordance with established guidelines 44  gRNA selection and preparation. Gene-specific guide RNAs were chosen using the GuideScan prediction tool 45 . The selected gRNA sequence was predicted to have no off targets with up to one base pair mismatch. crRNAs were synthesized by IDT as Alt-R CRISPR-Cas9 crRNAs. A bipartite synthetic gRNA was heteroduplexed using crRNAs and a tracrRNA according to IDT recommendations. Individual gRNAs were evaluated to ensure efficient induction of indels by microinjection (see description above) and by clonal analysis from a pool of injected embryos. The gRNA used induced indels in a minimum of 80% of clones analyzed from a pool of 10 embryos.
Clonal analysis of gene editing. Genomic DNA was isolated from a pool of 20 embryos (24 h post-fertilization, or hpf) with DirectPCR Lysis Reagent (Viagen) and Proteinase K at 20 μg/ml (Qiagen). Samples were incubated at 55 °C for 60 min then 85 °C for 45 min. Genomic DNA was PCR amplified using primers outlined in Supplementary Table S1. 8 μl PCR product was mixed with 1.6 μl NEB Buffer 2 and 6.4 μl of water and was hybridized by incubation at 95 °C for 5 min then cooled from 95-85 °C at − 2 °C/s and 85-25 °C at − 0.1 °C/s. The hybridized DNA digested for 1 h at 37 °C with 2 U of T7EI endonuclease (New England BioLabs (NEB). Digested product was visualized on a 2% agarose gel.
To perform clonal analysis, the PCR product was cloned into pCRII-TOPO (Invitrogen) and subjected to Sanger sequencing (Genewiz). Sequences were compared to the danio rerio reference genome and uninjected controls to identify indels with the MacVector software (Version 16.0).

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints.
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